Recombinant immunoglobulins and fusion proteins constructed using constant domain fragments of immunoglobulins are used to treat many human diseases including inflammatory diseases (e.g. rheumatoid arthritis, psoriasis, inflammatory bowel disease), allergies (e.g. asthma), cancers (e.g. lymphoma, breast cancer, bowel cancer), infectious diseases (e.g. RSV infection), pain (e.g. osteoarthritic pain, cancer pain, lower back pain) and eye disease (e.g. age-related macular degeneration).
The molecular targets for therapy include cytokines and chemokines (e.g. interleukin-1 (IL-1), interleukin-5 (IL-5), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor), growth factors (e.g. nerve growth factor (NGF), vascular endothelial cell growth factor (VEGF), tumour necrosis factor (TNF)), cell surface receptors (e.g. HER-2, VEGFR, EGFR, CD20), cell surface-bound growth factors (e.g. unprocessed tumour necrosis factor), viruses (e.g. RSV) and components of the complement cascade (e.g. C5). Many other targets that have evidence for involvement in disease processes are known (e.g. as described in the IMGT/MAb-DB database Version 1.3.1 14 Dec. 2011, (www.imgt.org/mAb-DB/query).
Native immunoglobulins are produced as different major subtypes, including immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) or immunoglobulin E (IgE) and in response to infection these immunoglobulins play various roles in pathogen recognition by binding to target antigens, neutralisation, destruction and removal. Immunoglobulin G is produced as several different isotypes (also known as isoforms), such as (in humans) IgG1, IgG2, IgG3 and IgG4. These antibody isotypes vary in structure, in particular with regard to differences in the amino acid sequences of the constant region, particularly around the hinge region of the constant domain (Fc) between the C1 and C2 domains.
Different antibody isotypes also differ in terms of the downstream effector functions which the antibody mediates. For example, the constant region sequence of an antibody can mediate a strong influence on characteristics such as effector functions (ADCC, complement fixing and activation), pharmacokinetics, and physical properties of an antibody. Antibodies having different isotypes also differ in terms of their ability to bind to IgG Fc receptors on immune cells. In humans, IgG1 and IgG3 are active in recruiting complement to aid in target destruction by the cascade of complement enzymes in the blood (CDC: complement-dependent cytotoxicity), and similarly IgG1 and IgG3 bind Fc receptors on immune cells that target the bound antigen for destruction by antibody-mediated cellular cytotoxicity (ADCC). By contrast, IgG2 and IgG4 do not recruit complement or activate ADCC mediated attack and simply bind to the target antigen with high affinity to inhibit or neutralise its activity.
Recombinant immunoglobulins and fusion proteins made from the same are designed to take into account the activity of the Fc isotype when considering the target for disease intervention. For example, it is preferable when considering a therapeutic approach which aims to use antibodies for the targeted killing of human cancer cells to construct the recombinant immunoglobulin from IgG1 or IgG3 isotype Fc domains, as the use of these isotypes will drive immune mediated destructive mechanisms such as CDC and ADCC. By contrast, when targeting soluble mediators in the context of sensitive human tissues, the Fc domain is either omitted (e.g. in treatment of human age-related macular degeneration Fab fragments targeting VEGF are preferred), or is constructed using IgG2 or IgG4 Fc domains (e.g. targeting nerve growth factor in the context of neuropathic or inflammatory pain, or complement C5 in nephritis, psoriasis or rheumatoid arthritis). These considerations also apply to immunoglobulin fusion proteins, such as soluble TNF receptor Fc fusion proteins in the treatment of conditions such as rheumatoid arthritis, which are based on human IgG1 Fc therapeutics.
In canines and other species such as mice and horses, immunoglobulin isoforms also exist but have insufficient homology between one another to determine a priori which sequence will be active or inactive in inducing downstream effector functions such as CDC or ADCC. Furthermore, the number of immunoglobulins varies between species (e.g. in dog there are four IgG immunoglobulins, these being defined as calgG-A, calgG-B, calgG-C, and calgG-D (Tang et al., 2001). In horses, there are seven IgG isotypes (Wagner, 2006).
It is not possible to determine from sequence analysis or sequence homology alone whether a specific immunoglobulin isotype of a non-human species will be active or inactive in terms of mediating Fc receptor binding and downstream effector function. However, if these were known, it would be of significant value as the choice of isotype constant regions for antibody generation can be critical in order to provide the therapeutic effectiveness of an antibody or antibody based therapeutics, such as an antibody binding fragment or fusion protein.